Human cells contain three billion base pairs of DNA, our genome, compacted into chromatin. Expression levels of proteins are controlled by chromatin structure through chemical modifications that provide a second layer of control beyond the DNA blueprint, collectively termed the epigenome. Alterations to the epigenome by methylation of histones and DNA are tightly coupled to many diseases, particularly cancer. At present, our ability to control the methylation state of histones as well as other proteins in the human genome is severely limited by the paucity of selective drugs that target histone methyl transferases (hMTases). An ever increasing body of knowledge suggests that hMTases are a druggable target class with considerable interest from both industry and academia. The ability to provide a comprehensive panel of direct assays for profiling inhibitors against the >50 hMTases will accelerate the development of new drug leads and new opportunities to dissect the role of epigenetic change in human disease. The overall goal of the proposed research is to develop high throughput assays with high signal to background that allow for comprehensive profiling of the enzymes responsible for protein methylation. The proposed goals will be addressed via two Specific Aims that build on our expertise in split-protein assays with luciferase. We have previously applied the three-hybrid methodology for the development of a 120 kinase assay panel for profiling inhibitors against the protein kinome that has been recently commercialized. In this proposal, Aim 1 will involve the development of a three hybrid cell-free assay for HMTases and demonstrate the generality of the assay with lysine and arginine methyltransferases that have already been the focus of drug development efforts. Specifically, we will develop three-hybrid assays for the lysine MTases G9a (EHMT2), GLP (G9a-like protein, EHMT1), and the arginine methyltransferases. CARM1. Aim 2 will focus on the development of three hybrid assays for interrogating methyltransferases that only function in the context of a complex with their cellular binding partners. Specifically, we will reconstitute the PRC2 complex responsible for H3K27 trimethylation, which has been implicated as a driver of oncogenesis and test for inhibition using our three-hybrid assay. In terms of significance, the results of the proposed research will provide the basis for the development of novel yet economical, high throughput methods for profiling inhibitors against human methyltransferase by both industry and academia. This will lead to the development of new drugs and tools for probing the methylation status of the epigenome controlled by particular MTases that are implicated in cancer and other diseases.